The embryonic stem cell test (EST) has recently been utilized as an alternative test for developmental toxicity despite its numerous improvements across a wide range of species, many issues still remain.However, all commercial available mouse embryonic stem cells (ESC) are with XY karyotype, such as D3 and E14TG2A;up to date, mouse ESC with XX karyotype have not been used in the EST due to its rarity, which restricts the ability of the mouse EST to identify potential gender-specific effect during high-throughput-screening (HTS) of chemicals.